Facioscapulohumeral muscular dystrophy (FSHD) Is one of the most common adult muscular dystrophies. Deletion of a subset of subtelomeric macrosatellite repeats (D4Z4 repeats) causes most cases of FSHD and results in a dominantly inherited disease. Recent work from our groups have shown a decrease of repressive epigenetic markings on the deleted allele, increased binding of regulatory factors to the deleted allele, and a complex set of coding and non-coding RNAs generated from the D4Z4 repeats. These findings strongly support a broad hypothesis for the pathophysiology of FSHD: loss of epigenetic silencing in the D4Z4 repeat leads to increased expression of an RNA species that causes the disease, either through an RNA or protein mediated mechanism. Our research groups will address specific and complementary aims that together will both test the broad hypothesis and identify the specific mechanism(s) of FSHD, which is critical for future therapy development. The aims of Project 1 (Dr. van der Maarel) will identify the DNA sequences and epigenetic changes specific to 4qA161 that are necessary to confer FSHD pathology. The aims of Project 2 (Dr. Tapscott) will determine whether the D4Z4-generated coding and non-coding RNAs have a role in FSHD pathology and identify mechanisms of generally suppressing transcription from the D4Z4 repeat region. The aims of Project 3 (Dr. Filippova) will be to determine whether CTCF binding on the disease associated allele regulates regional chromatin structure, RNA transcription, or nuclear localization. The aims of Project 4 (Dr. Miller) will be to use FSHD-derived IPS cells to identify the transcriptional an epigenetic determinants of FSHD, and the regulatory regions in the D4Z4 region. Dr. Tawil (Core A) has established an FSHD Bioresources Core with uniformly collected and well-characterized biological samples from FSHD and control individuals. The aims of Core A will be to collect DNA from blood samples for genetic analysis, fibroblast and myoblasts from skin and muscle for biological studies, and muscle biopsies for histochemical and in situ biological analyses. The Administrative Core (Core B) will coordinate the administration of the grant, communications among the participants, and reporting responsibilities. Together, these studies combine genetic, epigenetic, transcriptional and developmental approaches to defining the molecular deficits that cause FSHD and will provide a new basis for developing therapies. PUBLIC HEALTH RELEVANCE: The pathophsiology of FSHD is currently unknown and there are no validated targets or mechanisms that can provide the basis for therapeutic development. The significance of this proposed study is that it will identify and validate specific mechanisms for the pathophysiology of FSHD. The human health relevance of this work is that it will provide a new basis for rational therapeutic development for FSHD. PROJECT 1 Principal Investigator: Silvere van der Maarel, PhD Title: The Genetic and Epigenetic Basis for FSHD Description (provided by applicant): We have demonstrated that FSHD is caused by a contraction-cfepencfenf (FSHD1) or contractionindependent (FSHD2) change in chromatin structure of D4Z4 only when this contraction occurs on a specific genetic background (4qA161). This leads to the hypothesis that a change in D4Z4 chromatin structure on the 4qA161 haplotype is essential for FSHD pathology. Therefore, the long-term goal is to identify the specific DNA sequences and the epigenetic modifications that together confer pathogenicity to 4qA161. Aim 1 will identify and functionally characterize the disease haplotype-specific sequence variants of the distal repeat unit and flanking pLAM sequence. Recent studies identified this part of the FSHD locus as the minimal essential region;Aim 2 will identify and functionally characterize the chromatin structure of this minimal essential region test the hypothesis that the D4Z4 repeats regulate DUX4 expression and have a biological role in early embryonic development;and Aim 3 will determine the genetic and epigenetic characteristics of D4Z4 in human ES cells to establish the developmental role of D4Z4 in relation to the clinical features of FSHD. Public Health Relevance: The significance of these studies is that identifying the genetic and epigenetic conditions required for FSHD will provide fundamental insight into the pathophysiology of FSHD as well as providing new avenues for interventional therapies.